US4929444A - Low pH pharmaceutical formulation containing t-PA - Google Patents

Low pH pharmaceutical formulation containing t-PA Download PDF

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US4929444A
US4929444A US06/862,817 US86281786A US4929444A US 4929444 A US4929444 A US 4929444A US 86281786 A US86281786 A US 86281786A US 4929444 A US4929444 A US 4929444A
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formulation according
pharmaceutical formulation
aqueous solution
solution
concentration
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Michael D. Johnston
Henry Berger
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SmithKline Beecham Corp
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Burroughs Wellcome Co USA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to tissue plasminogen activator and in particular to pharmaceutical formulations containing tissue plasminogen activator, their preparation, and their use in human and veterinary medicine.
  • Blood clots consist of a fibrous network that is capable of dissolution by the proteolytic enzyme, plasmin.
  • the enzyme is derived from the inactive proenzyme, plasminogen, a component of blood plasma, by the action of a plasminogen activator.
  • plasminogen activator There are two immunologically distinct mammalian plasminogen activators.
  • Intrinsic plasminogen activator also known as urokinase, is an enzyme produced by the kidney and can be isolated from urine. It can also be prepared from a number of tissue culture sources.
  • Extrinsic plasminogen activator also known as vascular plasminogen activator and as tissue plasminogen activator (t-PA)
  • tissue plasminogen activator can be isolated from many tissue homogenates (notably human uterus), the vascular cell wall and from some cell cultures.
  • tissue homogenates notably human uterus
  • streptokinase prepared from beta-haemolytic streptococci.
  • streptokinase prepared from beta-haemolytic streptococci.
  • t-PA can, for example, destroy other blood proteins, such as fibrinogen, prothrombin, factor V and factor VIII so reducing blood clotting ability and increasing the risk of haemorrhage.
  • fibrinogen such as fibrinogen, prothrombin, factor V and factor VIII
  • t-PA the biological activity of t-PA is dependent on the present of fibrin to which it binds and where it is activated. Maximum activity is thus developed only at the site of a blood clot, i.e. in the presence of the fibrin network to be dissolved, and this greatly avoids the risk of haemorrhage.
  • t-PA The main route of administration of t-PA is by intravascular infusion, thus requiring the formulation of t-PA as a parenteral solution.
  • the drug In the case of a protein it is preferable to present the drug to the physician or veterinarian as a lyophilised pharmaceutical formulation because of its significant transportation and storage advantages over a liquid formulation. It is, however, important that any such lyophilised formulation is readily convertible into the desired parenteral solution without undue inconvenience and difficulty and that the physician or veterinarian is able to obtain the required concentration of drug in any given situation simply by reconstitution of the formulation in the appropriate amount of solvent. It is, for example, inadvisable to administer a large volume of solution to a patient with a cardiac or renal disorder since it would put the heart or kidneys under even greater stress. The volume should therefore be kept to a minimum in such circumstances. It is thus desirable that a parenteral solution can be obtained not only with a relatively low concentration but also with a high concentration of the drug.
  • lyophilised pharmaceutical formulations of t-PA have been described in the prior art, for example in EP-A-113 319, and EP-A-123 304.
  • the formulations are prepared from aqueous saline solutions of t-PA, in which the pH is about neutral, and suffer from the disadvantage that the solubility of t-PA in such solutions is low in the absence of an increase in the ionic concentration. Consequently, the parenteral solutions obtained from such lyophilised formulations either contain low concentrations of t-PA, necessitating in some situations the adminsitration of undesiably large volumes of solution to a patient, or thery are hypertonic, which on administration may be detrimental to red blood cells.
  • the present invention provides a process for preparing a lyophilised pharmaceutical formulation of t-PA, which comprises vacuum drying a frozen aqueous solution of t-PA, in which the pH is from 2 to 5.
  • a lyophilised pharmaceutical formulation can be prepared in accordance with the present invention that is capable of affording a parenteral solution containing a high concentration of t-PA without any substantial risk of the t-PA being precipitated out of solution.
  • the lyophilised formulation may, therefore, be presented to physicians or veterinarians who can dissolve the formulation, as and when required, to the desired concentration using, for example, water of neutral or acidic pH.
  • the present invention therefore, provides a stable lyophilised pharmaceutical formulation that allows for greater flexibility in its handling and use by physicians and veterinarians, as well as providing for a more convenient means of transportation and storage.
  • the t-PA of use with the present invention may be any bioactive protein substantially corresponding to mammalian, and especially human, t-PA and includes forms with and without glycosylation. It may be one- or two-chain t-PA, or a mixture thereof, as described in EP-A-112 122 and, in the case of fully glycosylated human t-PA, has an apparent molecular weight on polyacrylamide gels of about 70,000 and an isoelectric point of between 7.5 and 8.0.
  • the t-PA has a specific activity of about 500,000 IU/mg (International Units/ml, the International Unit being a unit of activity as defined by WHO, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London, NW3 6RB, U.K.).
  • the amino acid sequence of t-PA preferably substantially corresponds to that set forth in FIG. 1.
  • the sequence is thus identical to that in FIG. 1 or contains one or more amino acid deletions, substitutions, insertions, inversions or additions of allelic origin or otherwise, the resulting sequence having at least 80% and preferably 90%, homology with the sequence in FIG. 1 and retaining essentially the same biological and immunological properties of the protein.
  • the t-PA sequence is identical to that in FIG. 1 or has the same sequence but with the amino acid in the 245th position from the serine N-terminus being valine instead of methionine, either sequence optionally being without any of the first three amino acids or optionally having an additional polypeptide N-terminal presequence of Gly-Ala-Arg.
  • the amino acid sequence set forth in FIG. 1 has thirty-five cysteine residues and thus the potential for forming seventeen disulphide bridges.
  • the postulated structure for the sequence (arising from disulphide bond formation) between the amino acid in the 90th position and the proline C-terminus is set forth in FIG. 2.
  • the structure of the N-terminal region is less certain although some proposals have been put forward (Progress in Fibrinolysis, 1983, 6, 269-273; and Proc. Natl. Acad. Sci., 1984, 81, 5355-5359).
  • t-PA The most important features of the structure of t-PA are the two kringle regions (between the 92nd and the 173rd amino acids and between the 180th and 261st amino acids), which are responsible for the binding of the protein to fibrin, and the serine protease region, which comprises the major part of the B-chain and which is responsible for the activiation of plasminogen.
  • the amino acids of special significance in serine proteases are the catalytic triad, His/Asp/Ser. In t-PA these occur at the 322 nd, the 371st and the 463rd positions.
  • the disulphide bridge between the 264th and 395th cysteine amino acid residues is also important in that it holds together the A- and the B-chains in the two-chain form of t-PA.
  • the t-PA may be obtained by any of the procedures described or known in the art. For example, it may be obtained from a normal or neoplastic cell line of the kind described in Biochimica et Biophysica Acta, 1979, 580, 140-153; EP-A-41 766 or EP-A-113 319. It is preferred, however, that t-PA is obtained from a cultured transformed or transfected cell line, derived using recombinant DNA technology as described in, for example, EP-A-93 619; EP-A-117 059 or EP-A-117 060.
  • CHO cells Chinese hamster ovary (CHO) cells are used for the production of t-PA and are derived in the manner as described in Molecular and Cellular Biology, 1985, 5(7), 1750-1759.
  • the cloned gene is cotransfected with the gene encoding dihydrofolate reductase (dhfr) into dhfr - CHO cells.
  • Transformants expressing dhfr are selected on media lacking nucleosides and are exposed to increasing concentrations of methotrexate.
  • the dhfr and t-PA genes are thus coamplified leading to a stable cell line capable of expressing high levels of t-PA.
  • the t-PA is, preferably, purified using any of the procedures described or known in the art, such as the procedures described in Biochimica et Biophysica Acta, 1979, 580, 140-153; J. Biol. Chem., 1979, 254(6), 1998-2003; ibid, 1981, 256(13), 7035-7041; Eur. J. Biochem., 1983, 132, 681-686; EP-A-41 766; EP-A-113 319 or GB-A-2 122 219.
  • t-PA solubility of t-PA in the aqueous solution of use in the process of the present invention.
  • concentration of t-PA in the aqueous solution may vary therefore within wide limits, for example from 50,000 to 50,000,000 IU/ml.
  • concentration of t-PA is greater than 100,000 IU/ml, more especially greater than 500,000 IU/ml, and most especially greater than 1,000,000 IU/ml. It is most particularly preferred that the concentration of t-PA is about 5,000,000 IU/ml.
  • the upper limit of the pH of the aqueous solution is, preferably, 4.5.
  • the pH is, preferably, within the range from 2.5 to 4.0, more preferably from 2.8 to 3.5, and most preferably about 3.0.
  • the desired pH of the aqueous solution is conveniently obtained using a physiologically acceptable inorganic or organic acid.
  • examples of such an acid include hydrochloric acid, sulphuric acid and nitric acid, and citric acid, tartaric acid and benzenesulphonic acid. Of these examples, hydrochloric acid is preferred.
  • the medium for the aqueous solution is wholly or substantially aqueous.
  • the parenteral solution obtained from the lyophilised pharmaceutical formulation may be hypertonic, hypotonic or isotonic with the blood serum of the patient. To avoid undesirable side effects, however, the parenteral solution is, preferably, isotonic although minor deviations are not of great physiological concern.
  • a substantially isotonic parenteral solution may be obtained by the inclusion of a physiologically acceptable agent that is capable of raising the tonicity of the solution to the required level. The agent may be included within the aqueous solution to be freeze-dried so that it is already present in the lyophilised pharmaceutical formulation or it may be included within the water of neutral or acidic pH that is used to dissolve the formulation to obtain the desired parenteral solution.
  • an agent examples include dextrose (in anhydrous or monohydrate form) and sodium chloride and mixtures thereof.
  • concentration of the agent in the aqueous solution or in the water for dissolution will, of course, vary from agent to agent.
  • sodium chloride the concentration is perferably from 7 to 10 mg/ml, and most preferably about 8.5 mg/ml, the concentration often referred to as physiological saline solution or just physiological saline.
  • the concentration is preferably from 30 to 70 mg/ml, and most preferably about 50 mg/ml.
  • the aqueous solution may optionally contain additives normally associated with lyophilised pharmaceutical formulations of this type.
  • additives normally associated with lyophilised pharmaceutical formulations of this type include human serum albumin, and binders and fillers, such as mannitol, lactose and glucose.
  • t-PA has a tendency to adsorb to glass and plastic surfaces and, therefore, it may be desirable to include a surface active agent in the aqueous solution to prevent or minimise such adsorption.
  • examples of such an agent include polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides, such as that marketed under the trade name "Tween 80".
  • any additional material such as lysine or ornithine or a salt thereof, for enhancing the solubility of t-PA.
  • the aqueous solution of t-PA may be prepared by obtaining a solution of purified t-PA and exchanging the medium for an aqueous medium having a pH from 2 to 5, or by dissolving purified t-PA in an aqueous medium having a pH from 2 to 5.
  • the purification of t-PA may involve as a final stage the elution of the protein from a chromatographic column as a solution containing a strong buffering agent.
  • the parenteral solution and hence the lyophilised pharmaceutical formulation and aqueous solution, does not contain a strong buffering agent and, therefore, a convenient means for effecting its removal whilst exchanging the medium is to use dialysis.
  • This may be carried out using dialysis tubing or an artifical kidney in which the purified solution is dialysed against an aqueous medium in which the pH is from 2 to 5. It may be desirable, especially if the concentration of t-PA in the purified solution is high, first to adjust the pH of the solution so that it is from 2 to 5.
  • Another means for effecting the removal of a strong buffering agent whilst exchanging the medium is to subject the purified solution to gel filtration and to develop the column with an aqueous medium in which the pH is from 2 to 5.
  • t-PA in the form of a precipitated solid may, preferably, be obtained from a purified solution by adjusting the pH to about 5.5, cooling the solution to just above its freezing point, and recovering the protein by, for example, centrifugation.
  • the precipitated solid may then be dissolved in an aqueous medium having a pH from 2 to 5 in a conventional manner.
  • the resulting aqueous solution is sterilized conventionally, for example, by filter sterilization, and that it is then dispensed into sterile, plastic or glass containers, such as ampoules or vials, in volumes of, for example, from 0.5 to 20 ml.
  • the aqueous solution of t-PA is frozen, preferably, at a temperature of from -10° to -40° C.
  • the frozen aqueous solution is then, preferably, maintained at this temperaure until the vacuum drying is commenced.
  • Vacuum drying of the frozen aqueous solution may be carried out conventionally and includes drying under a partial or complete vacuum, for example at from 0.01 to 0.1 Torr, for sufficient time to effect removal of substantially all the frozen liquid.
  • the temperature at which vacuum drying is carried out is usually from -30° to -40° C. at the beginning of the process so as to maintain the aqueous solution in a substantially or completely frozen form. As the process proceeds and the water is removed, the temperature may be gradually increased until it reaches room temperature. It is preferred, at the end of the process, to carry out the vacuum drying at room temperature or just above under a substantial vacuum of about 0.01 Torr in order to remove as much as possible of the last traces of water.
  • the moisture content of the resulting lyophilised pharmaceutical formulation is preferably less than 2.5%.
  • the present invention also provides a physiologically acceptable salt, especially a physiologically acceptable acid addition salt, such as the hydrochloride salt, of t-PA.
  • the use of the present invention enables for the first time a lyophilised pharmaceutical formulation to be obtained which is capable of affording a parenteral solution containing a high concentration of t-PA. Accordingly, the present invention also provides a lyophilised pharmaceutical formulation of t-PA, which, on dissolution in water, is capable of affording a concentration of t-PA greater than 100,000 IU/ml, more especially greater than 500,000 IU/ml, and most especially greater than 1,000,000 IU/ml.
  • the lyophilised pharmaceutical formulation obtained according to the process of the present invention is reconstituted in water of neutral or acidic pH. If the aqueous solution from which the lyophilised pharmaceutical formulation was obtained is substantially isotonic, then it is preferred that the water for reconstitution is also substantially isotonic.
  • the biological activity of t-PA in dissolving the fibrin network of blood clots has led to its utility in the treatment of thrombotic disorders (The Lancet, Nov. 7th 1981, 1018-1020; ibid., Apr. 13th 1985, 842-847; The New England Journal of Medicine, 1984, 310(10), 609-613; and ibid., 1985, 312(14), 932-936).
  • the present invention therefore, provides a method for the treatment of a thrombotic disorder in a mammal, which comprises the administration to the mammal of a parenteral solution of t-PA obtained from a lyophilised pharmaceutical formulation, as defined herein.
  • a lyophilised pharmaceutical formulation of t-PA as defined herein, for use in human or veterinary medicine, especially for use in the treatment of a thrombotic disorder.
  • thrombotic disorder particularly examples include myocardial infarction, deep vein thrombosis, pulmonary embolism and stroke.
  • Intravascular infusions are normally carried out with the parenteral solution contained within an infusion bag or bottle or within an electrically operated infusion syringe.
  • the solution may be delivered from the infusion bag or bottle to the patient by gravity feed or by the use of an infusion pump.
  • gravity feed infusion systems does not afford sufficient control over the rate of administration of the parenteral solution and, therefore, the use of an infusion pump is preferred especially with solutions containing relatively high concentrations of t-PA. More preferred, however, is the use of an electrically operated infusion syringe which offers even greater control over the rate of administration.
  • an effective amount of t-PA to treat a mammal with a thrombotic disorder will of course depend upon a number of factors including, for example, the age and weight of the mammal, the precise condition requiring treatment and its severity, the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian. It is likely, however, that an effective amount for lysing a coronary artery thrombus, for example, will generally be in the range from 150,000 to 450,000 IU/kg bodyweight of patient per hour. Thus, for a 70 kg adult human being, an effective amount per hour will generally be from 10,000,000 to 30,000,000 IU, especially about 20,000,000 IU, and this amount may be administered with or without a priming dose.
  • the dosage will be less for some thrombotic conditions, such as deep vein thrombosis and acute stroke, or for simply maintaining patency of an already reperfused coronary artery. In these situations, an effective amount will generally be from 7,000 to 36,000 IU/kg bodyweight of patient per hour.
  • t-PA obtained from a cultured transformed CHO cell line which as derived using the procedure of Molecular and Cellular Biology, 1985, 5(7), 1750-1759, was purified chromatographically and the t-PA collected as an aqueous solution containing 0.17M sodium citrate and 0.01% (w/v) Tween 80 at a pH of 5.5. The pH of the solution was adjusted to 3.0 with hydrochloric acid and the resulting solution concentrated by ultrafiltration using an H-10 Cartridge (Amicon Ltd., Upper Hill, Stonehouse, Gloucestershire, England).
  • the concentrated aqueous solution was further purified by applying it to a gel filtration column (Sephadex G-150; Pharmacia Biotechnology, Uppsala, Sweden) and eluting with 0.85% saline solution containing 0.01% (w/v) Tween 80 at a pH of 3.0.
  • a highly purified aqueous solution of t-PA was thus obtained which was concenrated once more using a disposable artifical kidney.
  • the t-PA was precipitated out of solution by increasing the pH to 5.5 with sodium hydroxide and maintaining the suspension at 4° C. for 2 hours.
  • the t-PA was recovered by centrifugation at 4000 ⁇ g for 30 minutes at 4° C.
  • the pellet of t-PA was redissolved in an aqueous solution of sodium chloride (0.85% (w/v)) containing 0.01% (w/v) Tween 80 and adjusted to pH 3.0 with hydrochloric acid.
  • the volume of saline solution used was that required to give a concentration of t-PA between 7,500,000 IU/ml and 10,000,000 IU/ml.
  • This solution of t-PA was diluted with further aqueous solution of sodium chloride (0.85% (w/v)) containing 0.01% (w/v) Tween 80 and adjusted to pH 3.0 with hydrochloric acid, and also with sufficient fo a solution of 10% (w/v) mannitol in the same acid saline solution to give final concentrations of 5,000,000 IU/ml of t-PA and 25 mg/ml of mannitol.
  • the resulting solution was filter sterilized and dispensed in volumes of 1 ml into glass vials which were frozen at -35° C. A vacuum was applied at 0.05 Torr. After about 24 hours, the temperature was gradually increased to 5° C. and maintained at this temperature for 16 hours. It was then increased again to 25° C. and the vacuum increased to 0.02 Torr for a further 24 hours, after which the vials were sealed under a partial vacuum of 600 Torr of dry nitrogen.
  • the thrombolytic efficacy of a parenteral solution obtained from the lyophilised formulation of t-PA of Example 1 was evaluated in an in vivo model of jugular vein thrombosis.
  • Example 1 The lyophilised formulation of Example 1 rapidly and completely dissolved in sterile isotonic saline adjusted to pH3.0 containing 0.01% Tween 80. A parenteral solution was thus provided for a 2 hour infusion of 500,000 IU/kg of t-PA. Infusion was via a cannula in the right femoral vein. Four New Zealand white rabbits were used in the study. After infusion the degree of thrombolysis was estimated.
  • the percentage thrombolysis was 28.9 ⁇ 4.1 thus demonstrating the thrombolytic effect of the parenteral solution obtained from the lyophilised formulation of Example 1. In addition, there were no adverse reactions observed with this solution.

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GB858513358A GB8513358D0 (en) 1985-05-28 1985-05-28 Formulation
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
US5409699A (en) * 1989-12-20 1995-04-25 Boehringer Mannheim Gmbh Compositions containing glycosylated molecules having human tissue type plasminogen activator enzymatic activity
EP0827751A2 (en) * 1996-09-06 1998-03-11 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Medicinal composition containing tissue plasminogen activator and nicotinamide
EP0869970A1 (en) * 1995-12-13 1998-10-14 The Children's Medical Center Corporation Endothelial cell proliferation inhibitor and method of use
US20030077682A1 (en) * 2001-09-07 2003-04-24 Hung Paul Porwen Human tissue urokinase type plasminogen activator formulation
US20030219426A1 (en) * 1995-10-23 2003-11-27 O'reilly Michael S. Therapeutic antiangiogenic compositions and methods
US20040091465A1 (en) * 2002-06-26 2004-05-13 Zachary Yim Therapeutic antiangiogenic compositions and methods
US20060163752A1 (en) * 2004-04-05 2006-07-27 Xingwu Wang Storage assembly
US7495089B2 (en) 1995-10-23 2009-02-24 The Children's Medical Center Corporation Therapeutic antiangiogenic endostatin compositions
US20120040024A1 (en) * 2005-04-20 2012-02-16 Fred Hutchison Cancer Research Center Methods and compositions for enhancing survivability of cells, tissues, organs and organisms

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL78937A (en) * 1985-05-28 1990-11-29 Wellcome Found Pharmaceutical compositions containing tissue-plasminogen activator
ZW14486A1 (en) * 1985-07-29 1986-10-22 Smithkline Beckman Corp Pharmaceutical dosage unit
US5034225A (en) * 1985-12-17 1991-07-23 Genentech Inc. Stabilized human tissue plasminogen activator compositions
US4777043A (en) * 1985-12-17 1988-10-11 Genentech, Inc. Stabilized human tissue plasminogen activator compositions
IE59895B1 (en) * 1986-05-12 1994-04-20 Wellcome Found Medicaments containing tissue plasminogen activator
US4976959A (en) * 1986-05-12 1990-12-11 Burroughs Wellcome Co. T-PA and SOD in limiting tissue damage
GB8619098D0 (en) * 1986-08-05 1986-09-17 Wellcome Found Combination
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US7179608B2 (en) 1995-10-23 2007-02-20 The Children's Medical Center Corporation Therapeutic antiangiogenic compositions and methods
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US6139838A (en) * 1996-09-06 2000-10-31 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Tissue plasminogen activator medicinal composition
US20030077682A1 (en) * 2001-09-07 2003-04-24 Hung Paul Porwen Human tissue urokinase type plasminogen activator formulation
US20040091465A1 (en) * 2002-06-26 2004-05-13 Zachary Yim Therapeutic antiangiogenic compositions and methods
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